1. Field of the Invention
Cortisol (i.e., 11.beta.,17.alpha.,21-trihydroxy-4-pregnene-3,20-dione) is a steroid present in serum and urine. For the measurement of the steroid, several radioimmunoassay (hereinafter RIA) methods have been proposed and reported in, e.g., Anal. Lett. 5 757 (1972), ibid. 5. 767 (1972), Journal of Analytical Endocrinology and Metabolism 35 219 (1972), G. E. Abraham: "Radioimmunoassay of Plasma Steroid Hormones" in Modern Methods of Steroid Analyses Chap. 21, Academic Press, New York and London (1973), The Japanese Journal of Nuclear Medicine 12 123 (1975) Clinica Chimica Acta, 66 319-330 (1976), Clinical and Crinology (Tokyo) 24, 339 (1976) and the like. At the 94th Meeting of Pharmaceutical Society of Japan (Sendai, 1974), Tsuji et al proposed to produce an antibody to cortisol by injecting a cortisol-protein conjugate, i.e., cortisol-6.alpha.-O-hemisuccinate-bovine serum albumin conjugate (hereinafter, cortisol-6.alpha.-O-HS-BSA) having the formula (A) ##STR2## into rabbits. This proposal is described in T. Nishina, A. Tsuji and D. K. Fukushima: Steroids 24 (6) 861 (1974). This type of antigen is thought to be excellent for the production of the specific antibody because all of the functional groups of cortisol (i.e., the hydroxy groups at the 11.beta.-, 17.alpha.- and 21-positions and the oxo groups at the 3- and 20-positions) remain free.
On the other hand, tritium(.sup.3 H)-labeled cortisol is excellent immunologically as a tracer in RIA, but the procedure to synthesize tritium-labeled cortisol for use as a tracer is very complicated. Further measurement by counting the radioactivity should be made with a liquid scintillation counter, so that the use of such a tracer is not practical in a clinic.
As a result, investigations have now been made to prepare the conjugated compound of cortisol-6.alpha.-O-hemisuccinate with tyrosine methyl ester (i.e., cortisol-6.alpha.-O-HS-TME) having the formula (B) ##STR3## and to label this compound with radioactive iodine for use as a tracer. However, when attempts were made to employ this tracer in RIA, it was found the correct standard curve was not obtained. As a result of subsequent investigations, it was found that the immunological property of the tracer were different from that of cortisol and this difference caused the inaccuracy in the standard curve. Because, two different antibodies are produced by immunization with cortisol-6.alpha.-O-hemisuccinate-BSA conjugate, i.e., an antibody to cortisol and an antibody to cortisol-6.alpha.-O-hemisuccinate (antibody to the bridge) (the hemisuccinyloxy group in formula (A) or formula (B) above is considered a "bridge"). While the immunoreaction of cortisol occurs only with the antibody to cortisol, the tracer, radioiodinated cortisol-6.alpha.-O-HS-TME, can react with the antibody to cortisol as well as with the antibody to the bridge (anti-cortisol-6.alpha.-O-hemisuccinate). Therefore, the standard curve of cortisol indicates the sum of the quantity of cortisol and the quantity of the resultant of the immunoreaction between the tracer and the antibody to the bridge. FIG. 2 and FIG. 3 of the accompanying drawings show the above relationship, in which tritium-labeled cortisol and cortisol-6.alpha.-O-HS-TME-.sup.125 I are used as a tracer, respectively. Based on the above findings, this invention has now been accomplished.